CN111065421B - Wound dressing containing cross-linked gelatin derivative particles - Google Patents

Abstract

A wound dressing comprising particles comprising a crosslinked gelatin derivative comprising a structure represented by the following formula (1), GltnNH-CHR¹R²(1) In the above formula, Gltn is a gelatin residue, R¹Is an alkyl group having 1 to 17 carbon atoms, R²Is a hydrogen atom or an alkyl group having 1 to 17 carbon atoms, and the particle size of the particles is 1 to 1000 μm.

Description

Wound dressing containing cross-linked gelatin derivative particles

technical field

The present invention relates to a wound dressing, in detail, to a wound dressing containing particles of cross-linked gelatin derivatives.

Background technique

Wound dressings are defined as "materials that cover/protect wounds: used to absorb exudates, inhibit bleeding or loss of body fluids, and protect wounds from rubbing, rubbing, drying, contamination, Management of Covering/Protecting Wound Materials" (Japanese Pharmaceutical Affairs Law, Classification of Medical Device Categories). As wound dressings, there are gauze, polyurethane foam, hydrogel, etc., and these are all in the form of cloth.

Wound dressings have also come to be used in endoscopic submucosal dissection (ESD: Endoscopic Submucosal Dissection), which is a treatment for early stage cancers of the stomach, large intestine, and the like. The walls of the stomach, colon, etc. are composed of three layers, the mucosa, the submucosa, and the muscularis, and cancer arises from the innermost layer, the mucosa. In ESD, the submucosa including the mucosal layer is peeled off from the lumen of the digestive tract using a gastroscope and a colonoscope, and the lesions are excised together. If the excised tissue is left untreated, it will shrink and cause adhesions, which can lead to complications such as gastrointestinal obstruction. Therefore, the tissue is covered with a biodegradable wound dressing.

The wound dressing for ESD is required to have biodegradability and the like in addition to shapeability, that is, flexibility along the shape of the wound. A polyglycolic acid nonwoven fabric (manufactured by Gunze Co., Ltd., NEOVEIL (registered trademark)) is currently used. Although this polyglycolic acid nonwoven fabric is excellent in biodegradability and the like, it does not have adhesiveness to tissue, so it is necessary to cover and fix the nonwoven fabric with a fibrin-based adhesive. However, since this adhesive is expensive and is a blood preparation, the risk of contamination cannot be completely eliminated. In addition, the nonwoven fabric is difficult to spread at the application site, and thus lacks formability at the site of complex shape. Furthermore, there is also concern that polyglycolic acid oligomers, which are biodegradable substances, may cause tissue inflammation.

Gelatin is used for various medical purposes because of its excellent biocompatibility and biodegradability. As the particle preparation thereof, an embolic agent for transarterial embolization (Patent Document 1) and a sustained-release carrier for a drug (Non-Patent Document 1) are known. In addition, chemically modified gelatin has also been developed for medical use. For example, there is known a tissue adhesive which exhibits high adhesion to wet tissue by bonding a hydrophobic group to gelatin via an imino group (Patent Document 2).

【Existing technical documents】

【Patent Literature】

Patent Document 1: Japanese Patent Laid-Open Publication No. 2010-83788

Patent Document 2: Japanese Patent Application Publication No. 5995128

【Non-patent literature】

Non-Patent Document 1: Biometrics Vol. 21, pp. 489-499, 2000

SUMMARY OF THE INVENTION

[Technical problems to be solved by the invention]

An object of the present invention is to provide a biodegradable wound dressing that can be used to conform to the shape of a wound site and exhibit high adhesion to a wet site.

【Technical solutions for solving technical problems】

That is, the present invention is a wound dressing containing particles comprising a cross-linked gelatin derivative,

The gelatin derivative contains a structure represented by the following formula (1),

GltnNH-CHR ¹ R ² (1)

In the above formula, Gltn is a gelatin residue, R ¹ is an alkyl group with 1-17 carbon atoms, R ² is a hydrogen atom or an alkyl group with 1-17 carbon atoms,

The particle size of the particles is 1 to 1000 μm.

【Invention effect】

Since the above-mentioned wound dressing contains micron-sized particles, even a wound with a complex shape can be applied in accordance with the shape by spraying or the like. In addition, the particles have an alkyl group and have high adhesion to wet tissues. Furthermore, the particles can be prepared without using a dispersion medium and a crosslinking agent, so that they can be applied to a tissue without fear of adverse effects on the tissue due to residual or decomposition of the dispersion medium and the crosslinking agent.

Description of drawings

Figure 1 is an electron microscope (SEM) image of gelatin derivative particles.

Fig. 2 is a graph showing the change in adhesive strength according to the application amount of gelatin derivative particles.

Figure 3 is a ¹³ C-NMR spectrum of a gelatin derivative.

Fig. 4 is the FT-IR spectrum of 2500-3500 cm ^-1 of gelatin derivatives.

FIG. 5 is an SEM image of gelatin derivative particles before heat treatment after spray drying.

Fig. 6 is a phase contrast microscope photograph obtained by changing the heat treatment time in the range of 20 minutes to 3 hours, and dispersing the particles after the heat treatment in water immediately.

FIG. 7 is a graph for studying the solubility of particles in physiological saline according to heat treatment time by an accelerated test in the presence of collagenase.

8 is a graph plotting the amount of amino groups consumed when particles obtained by spray drying of raw gelatin are heat-treated at 150° C. with respect to time.

FIG. 9 is a graph showing the adhesive strength of gelatin derivative particles.

FIG. 10 is a graph showing the contact angles of gelatin (untreated) and gelatin derivative (hydrophobized gelatin) particles with respect to water.

FIG. 11 is a graph showing the viability of RAW264 cells in the presence of gelatin derivative particles.

Fig. 12 is a graph showing the proliferation behavior of Caco-2 cells on a membrane formed of gelatin derivative particles.

FIG. 13 is a graph showing IL-6 production by RAW264 cells in the presence of gelatin derivative particles.

14 is a photograph (upper section) and a graph (lower section) showing the wound healing process when gelatin derivative particles are applied to a skin wound of a rat.

Fig. 15 is a photograph (upper row) and a graph showing the positive area (lower row) of the α-smooth muscle actin-positive portion in the tissue on the 7th day after gelatin derivative particles were applied to the skin wound of a rat.

16 is an SEM image of particles prepared by a mechanical pulverization method using pollock gelatin as gelatin.

17 is a graph showing the adhesive strength of pollock gelatin derivative particles prepared by a mechanical pulverization method.

Detailed ways

<Gelatin Derivatives>

In the present invention, the gelatin derivative includes a structure represented by the following formula (1).

GltnNH-CHR ¹ R ² (1)

In the above formula, "Gltn" is a gelatin residue, R ¹ is an alkyl group having 1 to 17 carbon atoms, and R ² is a hydrogen atom or an alkyl group having 1 to 17 carbon atoms. N is derived from the ε-amino group of lysine (Lys) and is the main component in gelatin. Preferably R ² is a hydrogen atom. The NH structure of the formula (1) can be detected, for example, from a band near 3300 cm ⁻¹ in an FT-IR spectrum. The FT-IR spectrum of the gelatin derivative of Example 7 is shown in FIG. 4 .

When R ² is an alkyl group having 1 to 17 carbon atoms, it may be the same as R ¹ or different from each other. The alkyl group may also have a branched chain. Examples of the alkyl group include methyl, ethyl, propyl, butyl, hexyl, octyl (or octyl), nonyl (or nonenyl), decyl, dodecyl ( or lauryl), tetradecyl (or myristyl), etc. Preferably, R ¹ is a straight-chain alkyl group having 1 to 11 carbon atoms, more preferably a straight-chain alkyl group having 1 to 9 carbon atoms, and R ² is a hydrogen atom.

The derivatization rate in the gelatin derivative is 20 to 80 mol %, preferably 30 to 70 mol %, in terms of mol % of the alkyl group-bonded imino group and the amino group content in the raw gelatin. In other words, the imino group/amino group (molar ratio) in the obtained gelatin derivative is 20/80 to 80/20, preferably 30/70 to 70/30. The derivatization rate can be determined by quantifying the amino group in the raw gelatin and the amount of the amino group after the alkyl group is bound by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), or by NMR or the like. identification and quantification.

The raw material gelatin may be any of natural, synthetic, fermented, or genetically recombined gelatin, and preferably, gelatin derived from animals such as pigs and cattle, and fish such as pollock is used. In addition, although any of acid-treated gelatin, alkali-treated gelatin, and genetically-modified gelatin may be used, alkali-treated gelatin is preferable, and low-endotoxin gelatin is more preferable. In addition, in the molecular weight range of the gelatin, the weight average molecular weight (Mw) is preferably 30,000 to 150,000, and more preferably 50,000 to 120,000. The molecular weight can be determined by gel permeation chromatography (GPC) according to a conventional method.

<particle>

FIG. 1 is an SEM image of an example (Example 7) of particles in the present invention. It can be seen that the particles are approximately spherical. The particle size is roughly in the order of microns, that is, 1 to 1000 μm, mostly 1 to 50 μm, and the mode particle size (mode diameter) is 10 to 20 μm. The predetermined particle size range can be selected by sieving or the like according to the application site and application means of the particles. From the viewpoint of transportability and handleability, it is preferably 1 to 200 μm, and more preferably 1 to 50 μm. In the present invention, the particle diameter is determined by SEM image analysis, but may be determined by a laser diffraction method or the like.

The particles can be obtained by thermally crosslinking the gelatin derivative solution after spray-drying the gelatin derivative solution, or after mechanically pulverizing the solid gelatin derivative after drying, preferably a spray-drying method. As described in the above-mentioned Non-Patent Document 1, a method in which an aqueous gelatin solution is added dropwise to olive oil, gelatinized as a water-in-oil emulsion, and the obtained particles are added to an aqueous solution of glutaraldehyde for chemical crosslinking can also be used. According to the spray drying method, since no dispersion medium or curing agent is used, there is no need to worry about toxicity due to their residue or decomposition, and there is no need to wash the dispersion medium, so there is no need to worry about the inter-particle formation during drying after washing. Coagulation is preferred in these respects.

Crosslinking of the gelatin derivative can be confirmed by dispersing the particles in water and observing the insolubility of the particles visually or with a phase contrast microscope. Figures 6a-e show that the particles (Example 7) obtained by spray drying are put into a vacuum dryer, and the heat treatment time is 20 minutes to 3 hours under reduced pressure (<10Pa) and 150°C. The range was changed, the treated particles were immediately dispersed in water, and the micrograph was observed with a phase contrast microscope. Without heat treatment (Fig. 6a), when the treatment time was 20 minutes (Fig. 6b), the particles were dissolved in water, and when the treatment time was 1 hour (Fig. 6c), some undissolved particles were confirmed, and when the treatment time was 2 hours (Fig. 6d) A large number of particles were confirmed, and when the treatment time was 3 hours ( FIG. 6 e ), slightly more particles were confirmed than when the treatment time was 2 hours.

The solubility of the particles in the body fluid after the above-mentioned 1-hour, 3-hour and 6-hour heat treatment was investigated by an accelerated test in the presence of collagenase. 20 mg of each particle was dispersed in a 5 μg/mL collagenase/PBS (pH=7.4) solution, collected by centrifugation after 1, 2, and 4 hours, and the weight was measured after drying. The results are shown in FIG. 7 . As shown in this graph, by 3 hours of heat treatment, it is possible to show the desired initial absorption of body fluid without dissolving in body fluid when used as a wound dressing. In addition, it was found that as the heat treatment time increased, the dissolution time became longer, and the solubility in body fluids could be controlled by the heat treatment time.

Regarding the degree of crosslinking, the number of amino groups that can participate in the crosslinking reaction is different depending on the derivatization rate, and the degree of crosslinking defined as the state in which all the amino groups have reacted is 100% different, so the range cannot be limited. 8 is a graph showing the amount of amino groups consumed by cross-linking by measuring the amount of residual amino groups when the particles obtained by spray-drying the raw gelatin were subjected to heat treatment at 150° C. by the TNBS method, and plotted against time. . As can be seen from this figure, at 150° C., the crosslinking proceeded rapidly before 3 hours, then gradually slowed down, and reached about 40% of the amount of crosslinkable amino groups at 6 hours, which was almost the maximum. In the case of a gelatin derivative, the amount of amino groups remaining but not derivatized is smaller, so the reactivity is considered to be lower, and it is considered that the amount of amino groups reaches the maximum at about 10 to 30% of the amount of amino groups by heat treatment for 3 hours. However, as shown in the particles of Example 7, even if the derivatization rate was as high as 75%, the 3-hour heat treatment showed no problem in being used as a wound dressing with solubility resistance, which is considered to be due to the presence of hydrophobic alkanes. base.

<Additives, etc.>

The wound dressing of the present invention may contain various additives in an amount that does not hinder the object of the present invention. As such additives, there are colorants and preservatives. In addition, various pharmaceuticals may be contained, for example, antithrombotics, antibiotics, various growth factors, and the like. The particles can be loaded with additives and pharmaceuticals, or bonded thereto.

<Preparation method of wound dressing>

The preparation method of the wound dressing includes [1] a preparation process of a gelatin derivative, [2] a granulation process of spray drying or mechanically pulverizing the solid gelatin derivative after drying, and [3] a heat treatment process. Next, each step will be described.

[1] Preparation process of gelatin derivative

(1) Preparation of raw gelatin aqueous solution

The gelatin of the original material is used in an amount of 5 to 50 wt/v %, and heated at 40 to 90° C. to dissolve in water or an aqueous solvent. As water, ultrapure water, deionized water, and distilled water can be used. As the aqueous solvent, a mixture of water and a water-soluble organic solvent can be used. As the aqueous solvent, alcohols, esters, etc. having 1 to 3 carbon atoms can be used, and ethanol is preferably used.

(2) Derivative

To the gelatin solution obtained in the step (1), a derivatizing agent having an introduced alkyl group is added, and the mixture is stirred and reacted for a predetermined time. As the derivatizing agent, an aldehyde or ketone having the alkyl group, for example, dodecanal, tetradecanal, and decylethyl ketone can be used. The reaction temperature is 30 to 80° C., and the reaction time is 0.5 to 12 hours. Usually, gelatin in which an alkyl group is bonded to an amino group of gelatin via a Schiff base (˜N=CR ¹ R ² ) can be obtained just by stirring. The amount of aldehyde used is 1 to 4 times the stoichiometric amount corresponding to the desired derivatization rate. More preferably, it is 1 to 2 times.

Next, this Schiff base is reduced to the structure of the above-mentioned formula (1). As the reducing agent, known reducing agents such as sodium cyanoborohydride (NaBH ₃ CN), sodium triacetoxyborohydride (NaBH(OAc) ₃ ), 2-picoline borane, and pyridine borane can be used. Among them, 2-picoline borane is preferable. Picoline borane is stable and can perform reductive amination of aldehydes or ketones in one step (one-pot) in aqueous solvents. Also, a yield of 80 to 90% can be achieved. The amount of 2-picoline borane used is preferably 1 to 3 equivalents based on the equivalent of the derivative drug. In addition, the order of adding a reducing agent, an aldehyde, etc. may be arbitrary, and either one may be added to the gelatin solution first, or may be added simultaneously.

(3) Purification

To the reaction solution obtained in step (2), a large excess of a poor solvent such as cold ethanol is added, or the reaction solution is added to cold ethanol to precipitate the gelatin derivative. After filtering the precipitate, washing with ethanol or the like is performed to obtain the final product.

[2] Granulation process

＜Spray drying method＞

It has been found that the shape, size, etc. of the particles depend on many variable factors such as airflow velocity. Mainly from the observation of operability as a wound dressing, in order to obtain a large amount of particles of several tens of microns as large as possible within the above-mentioned particle size range, in addition to the molecular weight of the raw gelatin that has been described, the concentration of gelatin derivatives, Drying temperature, airflow velocity, solution flow velocity, etc. were studied. Next, each factor will be described.

The spray dryer may be any of a pan type, a nozzle type, a cyclone type, a filter type, and the like.

Spray drying is performed by dissolving a gelatin derivative in a mixed solvent of water and a water-compatible organic solvent, and spraying it under an inert gas such as nitrogen. As the organic solvent, alcohols, esters, etc. having 1 to 3 carbon atoms can be used, and ethanol is preferably used. The mixing ratio (volume) of water:organic solvent is 10:0 to 3:7, preferably 6:4 to 4:6. Although an aqueous gelatin solution is used in the method described in Patent Document 1, in the case of the gelatin derivative of the present invention, it is difficult to form particles using an aqueous solution, and sufficient adhesiveness cannot be obtained. As a result of various investigations, it was found that particles having excellent adhesion to a structure can be obtained by using the mixed solvent. Although the gist of the present invention is not limited, it is considered that the use of an organic solvent increases the evaporation rate of the solvent and aggregates the alkyl groups in the gelatin derivative at the gas-liquid interface, so that the alkyl groups appear on the surface of the spheres.

In this solution, water is added to the gelatin derivative, and the gelatin derivative is dissolved with gentle stirring at 50 to 90° C. An organic solvent is added to the obtained aqueous solution so that the concentration is 1 to 7w/v%, preferably 3 to 5w. /v%. If the concentration in the solution is less than the lower limit value, it is difficult to obtain particles, and even if it is greater than the upper limit value, the particles will adhere to the glass wall of the spray dryer before reaching the recovery container, and it is impossible to obtain a yield matching the concentration. improve.

The drying temperature is 140°C to 220°C, preferably 160°C to 200°C. When the temperature is less than the lower limit value, the particle size tends to be less than 1 μm, and when the temperature exceeds the upper limit value, the particles tend to aggregate.

The flow rate of the inert gas is 400 to 500 L/h, preferably 400 to 480 L/h. In addition, the flow rate of the gelatin derivative solution is 300 to 500 mL/h, preferably 350 to 450 mL/h.

<Mechanical crushing method>

The purified gelatin derivative is dissolved in ultrapure water to be in a solution state, dried at 40 to 60° C. to form a solid state, and then a fine pulverizer such as a roll mill, jet mill, or hammer mill is used. etc. to be crushed. When making it into a solution state, you may add a dispersion medium, such as polyethylene glycol.

[3] Heat treatment process

The particles obtained by spray drying or mechanical pulverization are heat-treated to crosslink the gelatin derivative. The heating temperature and time need to be defined and adjusted according to the molecular weight of the raw gelatin, the derivatization rate, and the target degree of crosslinking. For example, when the molecular weight (Mw) of the raw material gelatin is about 100,000, heating is performed at 140 to 160° C. for at least 3 hours.

After the heat treatment, the particles may be washed, dried, sieved, and treated to carry a chemical or the like.

<Approach to Organization>

The wound dressing of the present invention can be applied to incisions, skin wounds, etc. in various surgical operations such as respiratory surgery, especially wounds after lung cancer surgery, digestive surgery, cardiovascular surgery, oral surgery, gastroenterology and the like. In the case of ESD, it can be applied in a dry state through hemostatic forceps, stents, balloons, endoscopes, and the like. The application amount can be appropriately adjusted depending on the application site and wound. From the viewpoint of the adhesive strength to the tissue, by the same method as used in the examples described later, the amount of particles applied to the porcine stomach inner wall tissue was changed in the range of 1.6 to 32 mg/cm ² . As a result of measurement of the adhesive strength, it was found that it is preferably 10 to 20 mg/cm ² per unit area of the structure ( FIG. 2 ).

Example

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited thereto.

<Preparation of Gelatin Derivatives>

The gelatin derivatives shown in Table 1 were prepared. As an example, the preparation of gelatin derivative 7 is described.

5 g of pork skin-derived alkali-treated gelatin (Mw=100000, beMatrix (trade name), manufactured by Nitta Gelatin Co., Ltd.) was added to 50 mL of ultrapure water in an eggplant-shaped flask immersed in an oil bath at 50°C , which was dissolved while stirring for about 1 hour to prepare a 10 wt % aqueous solution. To the obtained aqueous solution, 777 mg of picoline borane (equivalent to about 1.5 times the equivalent of octanal, manufactured by Junsei Chemical Co., Ltd.) dissolved in 5 mL of ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added, followed by about 621 mg of octanal (manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in 70 mL of ethanol was added dropwise at a rate of 100 mL/h. The 621 mg was equivalent to about 3 times the target derivatization rate of 75 mol% for the amino group of gelatin. A reflux condenser was attached to the eggplant-shaped flask, and after reacting at 50°C for 17 hours while stirring, the eggplant-shaped flask was taken out from the oil bath, and ethanol was distilled off at 40°C using an evaporator. The obtained reaction solution was added dropwise to 1 L of ethanol in a beaker immersed in an ice bath to reprecipitate. After stirring for 1 hour, it was left to stand in a refrigerator for 1 hour, and then filtered with a glass filter. The filtration residue was dropped again into 1 L of ethanol in a beaker to reprecipitate, and after stirring for 1 hour, it was left to stand in a refrigerator for 1 hour. After filtration with a glass filter again, the filtration residue was dried in a vacuum desiccator overnight or more, and an octyl group-introduced gelatin derivative was obtained with a yield of 83%.

The introduction rate of the octyl group in the obtained gelatin derivative was obtained by the following method. Raw material gelatin and gelatin derivatives were dissolved in a water-DMSO mixed solvent (volume ratio 1:1, the same below) at 0.1 w/v%, respectively, and 100 μL was dispensed into a 48-well plate. Thereto was added 100 μL of 0.1 v/v% triethylamine (TEA, manufactured by Nacalai Tesque Co., Ltd.) dissolved in a water-DMSO mixed solvent, and the mixture was stirred at 400 rpm for 1 minute with a plate shaker. Then, 100 μL of 0.1 w/v% trinitrobenzenesulfonic acid (TNBS, manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a water-DMSO mixed solvent was added, and the mixture was stirred at 400 rpm for 1 minute with a plate shaker. The light was shielded with aluminum foil and allowed to stand in an incubator at 37° C. for 2 hours, then taken out from the incubator, 50 μL of 6N HCl was added to stop the reaction, and the plate was stirred at 400 rpm for 1 minute with a plate shaker. After standing for 10 minutes with shielding, the absorbance (Abs) at 340 nm was measured with a spectrophotometer (manufactured by TECAN, Spark 10M-NMST). From the measured absorbance, the absorbance of blank samples differing only in that they did not contain gelatin was subtracted, and the octyl group introduction rate of the gelatin derivative was determined to be 75 mol% by the following calculation formula.

Import rate (%)=[Abs (raw gelatin)-Abs (gelatin derivative)]/[Abs (raw gelatin)]×100

¹³ C-NMR and FT-IR of the obtained gelatin derivative were measured. ¹³ C-NMR is a measurement performed at 50° C. by dissolving the gelatin derivative and the raw gelatin in heavy water (containing 3-(trimethylsilyl)-1-propanesulfonate sodium as an internal standard) at 20 wt%, respectively. . The obtained ¹³ C-NMR spectrum is shown in FIG. 3 . In this gelatin derivative, a primary carbon-derived peak was observed around 17 to 17.5 ppm, and it was confirmed that an octyl group was introduced into the gelatin. The FT-IR measurement is performed directly by placing the gelatin derivative powder on the sample stage. In FIG. 4 , a part of 2500 to 3500 cm ⁻¹ of the FT-IR spectrum is shown. A peak derived from stretching vibration of methyl group (2868 cm ^{−1 ), a peak derived from stretching vibration of methylene group (2929 cm −1 )} , and a peak derived from stretching vibration of NH (3299 cm ⁻¹ ) were confirmed.

Other gelatin derivatives were prepared in the same manner as the above-mentioned method except that acetaldehyde, butyraldehyde, hexanal, or undecaldehyde was used in place of octanal in an amount corresponding to the target derivatization rate. The yields were all 80-90%. In addition, in Table 1 and the like, for example, "75C8" represents gelatin substituted with a C8 aldehyde at a substitution rate of 75% (R ¹ is a heptyl group and R ² is a hydrogen atom in formula (1)).

[Table 1]

<Spray drying>

(1) The gelatin derivative was dissolved in ultrapure water at 50° C. to prepare a 6 wt % aqueous solution.

(2) The same volume of ethanol was added to the aqueous solution to prepare a 3 wt% solution.

(3) The temperature of the solution was maintained at 50°C, and a spray dryer device (mini spray dryer, B-290, manufactured by Buchi Corporation) was installed, and at 180°C, the flow rate of nitrogen was 440 L/h, and the solution was The particles were obtained by spray drying at a flow rate of 410 mL/h. SEM images of the particles obtained from gelatin derivative 7 before heat treatment after spray drying are shown in FIG. 5 . As a comparative example, underivatized gelatin was similarly spray-dried.

<Heat treatment>

In FIGS. 6 and 7 , according to the results of the study on the above-mentioned heat treatment, the particles were heat-treated at 150° C. for 3 hours.

The gelatin derivatives 1 to 6 and 8 and the raw material gelatin were also spray-dried and heat-treated (150° C., 3 hours) under the same conditions to prepare each particle.

An SEM image of the particles obtained from the gelatin derivative 7 is shown in FIG. 1 . The particles were substantially spherical, and as a result of image analysis, the particle size distribution was in the range of about 1 μm to 1000 μm, and the mode particle size was 10 to 20 μm.

<Measurement of Adhesion Strength of Particles to Pig Stomach Inner Tissue>

Adhesion tests were performed using the inner wall tissue of pig stomach (Shibaura viscera). The test method was performed according to the standard of American Society for Testing Materials (ASTM F-2258-05). The pig stomach was opened and the mucosal layer was removed. At this time, physiological saline is injected into the submucosal tissue, and by removing the raised portion, only the mucosal layer is excised with the submucosal tissue remaining to some extent. The obtained tissue was cut into tissue pieces of 2.5 cm square, and fixed on the upper and lower jigs of the test device using an instant adhesive. The temperature of the porcine stomach lining tissue in the assay was maintained at 37°C by using a hot plate. Excess moisture from the tissue surface was removed with a kimwipe, and 100 mg (16 mg/cm ² ) of gelatin particles were placed on the tissue. After being crimped by the upper jig at 80 kPa for 3 minutes, the adhesive force was measured by pulling upward. Particles obtained from raw gelatin were used as Comparative Example 1, and NEOVEIL (trademark) was used as Comparative Example 2. The control is the blank value without any sample. The results are shown in Table 2 and FIG. 9 .

[Table 2]

<Hemostatic effect>

1 mL of whole blood collected from a rat (Wistar, 6 weeks old, female) was placed in a 5 mL vial, 50 mg of the particles of Example 7 (75C8) were added thereto, mixed gently, and then allowed to stand. Whole blood without any additions was used as a control. After mixing for 3 minutes, the control did not coagulate, whereas blood coagulation was confirmed in the sample containing the 75C8 particles. This shows that the gelatin derivative particles of the present invention have a hemostatic effect.

<Evaluation of Hydrophobicity of Gelatin Derivative Particles by Contact Angle Measurement>

About 100 mg of each particle of Example 1 (C2), Example 2 (C4), Example 3 (C6), Example 6 (C8), Example 8 (C12) and Comparative Example 1 (ORG) were placed on the carrier. On the glass slide, another slide was placed from above and the particles were compressed by hand to make pellets. Using a contact angle measuring device (DM-700, KYOWA), a droplet of ultrapure water was dropped on the surface of the pellet, and the angle between the droplet and the substrate was measured after 200 seconds. The results are shown in FIG. 10 .

As shown in FIG. 10, Comparative Example 1 is a material having a hydrophilic surface with a contact angle of 46 degrees. On the other hand, in the gelatin particles of Examples, it was confirmed that the contact angle increases with the chain length of the modified alkyl group. increase and increase. In particular, from C6 to C12, hydrophobic surfaces with contact angles exceeding 100 degrees were observed. That is, it turned out that the particle|grains which have a more hydrophobic surface can be obtained by modifying with a more hydrophobic group.

<Cell Viability Evaluation>

Using mouse-derived RAW264 cells as macrophage-like cells, the effect of gelatin-derived particles on cell viability was evaluated. RAW264 cells ^were seeded at a density of 6x10 cells/cm in 96 ^- well plates in RPMI1640 medium (10% fetal bovine serum, 1% non-essential amino acids, 1% antibiotics) at 37 °C, 5% _CO The incubator was incubated for 24 hours. Example 1(C2), Example 2(C4), Example 3(C6), Example 6(C8), Example 1(C2), Example 2(C4), Example 3(C6), Example 6(C8), Each particle of Example 8 (C12) and Comparative Example 1 (ORG) was added to the cells at 100 μL, and cultured for 24 hours. Then, the number of cells was quantified by a cell number quantification kit (WST-8, Dojin Chemical). The number of cells in each sample is shown in a graph ( FIG. 11 ) when the number of cells in the well to which no particles have been added is taken as 100%.

In the samples to which the particles of Example 1 (C2), Example 2 (C4), Example 3 (C6), Example 6 (C8) and Comparative Example 1 (ORG) were added, showed in all concentration ranges High cell numbers were obtained, confirming high cytocompatibility. On the other hand, in the particles of Example 8 (C12), a decrease in the number of cells was observed at a concentration of 2.5 mg/mL or more. From this, it can be said that it is preferable to apply particles with a long alkyl chain to a wound at a relatively low concentration.

<Effect of gelatin derivative particles on cell proliferation>

Wound healing involves the process of epithelialization, the closure of the wound surface by newly proliferating epithelial cells. Therefore, in order to evaluate the influence of gelatin derivative particles on cell proliferation, the proliferation behavior of Caco-2 cells, which are derived from human colon cancer cell lines, on membranes formed of gelatin particles was evaluated. The particles of Example 1 (C2), Example 2 (C4), Example 3 (C6), Example 6 (C8), Example 8 (C12) and Comparative Example 1 (ORG) were prepared at 0.2 mg/mL Each concentration was suspended in ultrapure water, and after adding 100 μL to each 96-well plate, the ultrapure water was evaporated to prepare a film formed of particles. After the membrane was sterilized by UV irradiation for 1 hour, Caco-2 cells were seeded on the membrane at a density of 6× ^{10 3} cells/cm ² , and the membrane was incubated in DMEM medium (20% fetal bovine serum, 1% non-essential amino acids) , 1% antibiotics) in a 37°C, 5% CO2 incubator for 24 hours. Then, the number of cells was quantified by a cell number quantification kit (WST-8, Dojin Chemical). The results are shown in FIG. 12 .

As can be seen from Figure 12, cell proliferation was carried out on the membranes formed from the particles of Example 1 (C2), Example 2 (C4), Example 3 (C6) and Comparative Example 1 (ORG), especially in Example 2 The particles of (C4) and Example 3 (C6) exhibited significantly higher proliferative properties than Comparative Example 1, indicating that the epithelialization process can be promoted. On the other hand, on the films formed from the particles of Example 6 (C8) and Example 8 (C12), the proliferation of Caco-2 was inhibited. Considering the results of the above-described cell viability evaluation, this may be due to the influence of the particle density of the alkyl chains on the membrane.

<Effect of gelatin derivative particles on cytokine production>

Instead of quantifying the number of cells, the procedure was the same as that in the evaluation of cell viability, except that the supernatant after culturing for 24 hours was collected and the amount of mouse IL-6 production was quantified using an ELISA assay kit (R&Dsystems). steps to quantify mouse IL-6 production. The results are shown in FIG. 13 . In this figure, "CTR" represents the sample without added particles. As shown in FIG. 13 , in the presence of the particles of Example 6 (C8) and Example 8 (C12), production of IL-6 was observed, and particularly, a large amount of IL-6 was produced in the particles of Example 8 (C12). IL-6 is involved in fever, angiogenesis, etc. in the acute phase reaction, and has various physiological activities, among which the acute phase reaction is involved in recovery from an undesired inflammatory state. By using the particles of Example 6 (C8) and Example 8 (C12), it can be expected to promote wound healing.

<Wound Healing Effect>

In order to confirm the wound-healing-promoting effect of the present particle, animal experiments were conducted using rats. Under inhalation anesthesia with isoflurane, a full-thickness skin defect was made on the back of a rat (female, 7 weeks old) using an 8 mm dermapunch. 10 mg of the particles of Example 2 (C4), Example 3 (C6), Example 6 (C8), Example 8 (C12) and Comparative Example 1 (ORG) were coated thereon, respectively. As a control (CTR), samples without coated particles were also made. A silicone sheet was placed thereon and fixed with tape, thereby holding the sample stably. After 7 days and 14 days, photographs of the wound (upper section in FIG. 14 ) and area measurement (lower section in FIG. 14 ) were taken, and histological sections were observed for the samples after 7 days ( FIG. 15 ).

As shown in FIG. 14 , it was found that on day 7, the residual area of the wound was very large in all the examples, except for example 8 (C12), which had approximately the same degree of wound closure as the CTR. When the wound is healed, if the wound closes rapidly while the inflammatory reaction is not completed, scar contracture which also leads to constriction may be caused, so it is preferable that the residual area of the wound be large. By applying the particles of the present invention, it was shown that the residual area was increased, and scar contracture was suppressed. On day 14, wound closure was confirmed in all samples.

The inflammation grade was evaluated by observing the tissue section after 7 days. The wound tissue was recovered by killing the rats with an excess of pentobarbital. After paraffin embedding, thin sections were prepared, and α-smooth muscle actin (α-smooth muscle actin: α-SMA), a protein expressed by inflammation, was stained and observed. Quantification of the area of the α-SMA positive part was performed using image processing software (ImageJ). As shown in the graph in the lower part of FIG. 15 , it was found that the particles of all the examples suppressed the expression of α-SMA more than CTR, including Example 8 (C12), which was approximately the same level as CTR for wound closure. From these results, by using the particles of the present invention as wound dressings, it is expected that the inflammatory reaction can be suppressed and wound healing can be promoted.

<Preparation of particles by mechanical pulverization>

In place of the alkali-treated gelatin derived from pig skin, alkali-treated pollock gelatin (Mw = 30 kDa, manufactured by Nitta Gelatin Co., Ltd., hereinafter abbreviated as "pollin gelatin") was used, except that about 2 times the equivalent amount of pollock gelatin was used. Gelatin derivatives were synthesized in the same manner as in Examples 1 to 8 except for octanal. The introduction rate was 58% (58C8). The obtained gelatin derivative was dissolved in ultrapure water to prepare 20wt% and 30wt% solutions, put into a Teflon (registered trademark) tray, heated at 40°C and dried to obtain a solid gelatin derivative thing. At this time, in order to evaluate the presence or absence of a dispersion medium, a sample to which 1% polyethylene glycol (PEG, Mw=1500 Da) was added before air-drying was also prepared. The air-dried solid gelatin derivative was pulverized for 1 minute using a Wonder Crusher, and was repeated three times. Finally, thermal crosslinking treatment was performed at 150° C. for 3 hours under reduced pressure. The obtained particles were particles having a particle diameter of about 20 to 50 μm as shown in the SEM image ( FIG. 16 ). No significant difference in particle size due to the presence or absence of PEG was found.

In the same manner as in Example 1 and the like, the adhesive strength to the porcine stomach lining tissue was measured. The results are shown in FIG. 17 . According to the results of the adhesion test, pollock gelatin has an adhesion strength (15 to 20 kPa) equivalent to that of pig gelatin. In addition, it was found that particles having excellent adhesive strength can be obtained by drying and pulverizing treatment without using a dispersion medium.

Industrial Availability

The wound dressing of the present invention is very useful in applications to wounds after surgical operations such as endoscopic mucosal dissection, vascular anastomosis, and the like.

Claims (5)

1. a wound dressing, is characterized in that, containing particles comprising cross-linked gelatin derivatives, The gelatin derivative has a structure represented by the following formula (1), GltnNH-CHR ¹ R ² (1) In the above formula, Gltn is a gelatin residue, R ¹ is an alkyl group with 1-17 carbon atoms, R ² is a hydrogen atom or an alkyl group with 1-17 carbon atoms, The particles are substantially spherical, formed by spray drying, and have a particle size of 1 to 1000 μm, The spray drying is performed by spraying a solution obtained by dissolving the gelatin derivative in a mixed solvent of water and an organic solvent compatible with water under an inert gas. 2. The wound dressing of claim 1, wherein The gelatin is obtained by subjecting gelatin derived from pigs or pollock to alkali treatment. 3. The wound dressing of claim 2, wherein The gelatin is low endotoxinized gelatin. 4. The wound dressing according to any one of claims 1 to 3, wherein The imino group/amino group (molar ratio) in the gelatin derivative is 20/80 to 80/20. 5. a preparation method of wound dressing, is characterized in that, For the preparation of the wound dressing according to any one of claims 1 to 4, it comprises the following steps: [1] A step of producing a gelatin derivative having the structure represented by the above formula (1); [2] a step of spray-drying the solution of the gelatin derivative to thereby perform granulation; and [3] A heat treatment step of heating the granulated particles at 140 to 160° C. for at least 3 hours, The spray drying is performed by spraying a solution obtained by dissolving the gelatin derivative in a mixed solvent of water and an organic solvent compatible with water under an inert gas.

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